WO2019237995A1

WO2019237995A1 - Drug for simultaneously reducing risk of haemorrhage and microvascular obstruction

Info

Publication number: WO2019237995A1
Application number: PCT/CN2019/090431
Authority: WO
Inventors: 姜宝红; 王琳琳
Original assignee: 中国科学院上海药物研究所
Priority date: 2018-06-15
Filing date: 2019-06-06
Publication date: 2019-12-19
Also published as: CN110604734A

Abstract

Provided in the present invention is a drug for simultaneously reducing the risk of haemorrhage and microvascular obstruction. Specifically, the present invention provides a composition, the composition comprising (a) a therapeutically effective dose of a first active ingredient, the first active ingredient being clopidogrel or a pharmaceutically acceptable salt thereof; (b) a therapeutically effective dose of a second active ingredient, the second active ingredient being salvianolic acid B or a pharmaceutically acceptable salt thereof; and (c) a therapeutically effective dose of a third active ingredient, the third active ingredient being ginsenoside Rg1 or a pharmaceutically acceptable salt thereof. The first active ingredient, the second active ingredient, and the third active ingredient of the present invention can produce a bilateral synergistic protective effect, effectively reducing microvascular obstruction and effectively preventing haemorrhage, and reducing the dosage of single drugs and reducing drug toxicity.

Description

Drugs that simultaneously reduce the risk of bleeding and microvascular obstruction

Technical field

The present invention relates to the field of medicine, and in particular, the present invention relates to a medicine that simultaneously reduces the risk of bleeding and microvascular obstruction.

Background technique

Ischemic reperfusion injury (ischemia reperfusion injury) refers to the recovery of blood flow to tissues or organs after ischemia for a period of time. Tissue or organ damage has not been reduced, but has become aggravated. Ischemia-reperfusion injury of important organs or tissues such as the heart, brain, kidney, liver, and skeletal muscle is seriously endangering human life and health. Risks of bleeding and microvascular occlusion often occur during surgical procedures such as thrombolytic surgery or vascular stent surgery.

How to effectively prevent and reduce ischemia-reperfusion injury of various tissues and organs of the human body and improve bleeding and microvascular obstruction are the difficulties and hotspots of current basic medical and clinical medical research.

Therefore, there is an urgent need in the art to develop a medicament with high effects and low side effects on ischemia-reperfusion injury, bleeding and microvascular obstruction.

Summary of the Invention

The object of the present invention is to provide a medicament with high effect and low side effects on ischemia-reperfusion injury, bleeding and microvascular obstruction.

According to a first aspect of the present invention, a composition is provided, the composition comprising:

(a) a therapeutically effective amount of a first active ingredient, the first active ingredient being clopidogrel or a pharmaceutically acceptable salt thereof;

(b) a therapeutically effective amount of a second active ingredient, said second active ingredient is salvianolic acid B or a pharmaceutically acceptable salt thereof; and

(c) a therapeutically effective amount of a third active ingredient, the third active ingredient being ginsenoside Rg1 or a pharmaceutically acceptable salt thereof.

In another preferred example, the pharmaceutically acceptable salt is a sulfate, a hydrogen sulfate, or a hydrochloride.

In another preferred example, the active ingredient of the composition is (a) a therapeutically effective amount of a first active ingredient, the first active ingredient being clopidogrel or a pharmaceutically acceptable salt thereof; and (b) A therapeutically effective amount of a second active ingredient, said second active ingredient consisting of salvianolic acid B or a pharmaceutically acceptable salt thereof; and (c) a therapeutically effective amount of a third active ingredient, said third active ingredient being Composition of ginsenoside Rg1.

In another preferred example, the content of the first active ingredient ranges from 0.01% to 99.99%, based on the total weight of the active ingredients of the composition; preferably from 0.1% to 99.9%; more preferably from 1% to 99%; Preferably 10% to 99%; more preferably 20% to 99%.

In another preferred example, the content of the second active ingredient ranges from 0.01% to 99.99%, based on the total weight of the active ingredients of the composition; preferably 0.1% to 99.9%; more preferably 1% to 99%; more Preferably 1% to 90%; more preferably 1% to 80%.

In another preferred example, the content of the third active ingredient ranges from 0.01% to 99.99%, based on the total weight of the active ingredients of the composition; preferably from 0.1% to 99.9%; more preferably from 1% to 99% ; Preferably 10% to 99%; more preferably 20% to 99%.

In another preferred example, the weight ratio of the clopidogrel or its pharmaceutically acceptable salt, salvianolic acid B or its pharmaceutically acceptable salt, and ginsenoside Rg1 or its pharmaceutically acceptable salt is 0.5-8: 0.5-6: 0.6-12, preferably 1-6: 0.5-4: 1-10, more preferably 2-4: 1-3: 3-7.

In another preferred example, the weight ratio of the second active ingredient to the third active ingredient is 0.5-6: 0.6-12, preferably 0.5-4: 1-10, and more preferably 1-3: 3-7.

In another preferred example, the total ratio of the first active ingredient to the second active ingredient is 0.5-8: 0.5-9, preferably 0.5-8: 0.5-6, preferably 1-6: 0.5-4 , More preferably 2-4: 1-3.

In another preferred example, the total ratio of the first active ingredient to the third active ingredient is 0.5-8: 0.6-12, preferably 1-6: 1-10, more preferably 2-4: 3-7 .

In another preferred example, the purity of the clopidogrel or a pharmaceutically acceptable salt thereof is 80-100%, preferably 90% -100%.

In another preferred example, the purity of the salvianolic acid B or a pharmaceutically acceptable salt thereof is 80-100%, preferably 90% -100%.

In another preferred example, the purity of the ginsenoside Rg1 or a pharmaceutically acceptable salt thereof is 80-100%, preferably 90% -100%.

In another preferred example, the composition is a pharmaceutical composition.

In another preferred example, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

According to a second aspect of the present invention, a kit is provided, and the kit includes:

(A) a first formulation containing clopidogrel or a pharmaceutically acceptable salt thereof;

(B) a second formulation containing salvianolic acid B or a pharmaceutically acceptable salt thereof;

(C) a third formulation containing ginsenoside Rg1 or a pharmaceutically acceptable salt thereof; and

(D) Instruction manual.

In another preferred example, the first preparation, the second preparation and the third preparation are mutually independent preparations.

In another preferred example, at least one of the first preparation, the second preparation and the third preparation is an independent preparation.

In another preferred example, the first preparation, the second preparation and the third preparation are combined preparations.

In another preferred example, the first formulation is a solid formulation, preferably a tablet.

In another preferred example, the second preparation is a liquid preparation, preferably an injection, an infusion or a lyophilized preparation.

In another preferred example, the third preparation is a liquid preparation, preferably an injection, an infusion or a lyophilized preparation.

In another preferred example, the instructions for use indicate that the first preparation, the second preparation and the third preparation are used in combination to prevent and / or treat ischemia-reperfusion injury, reduce bleeding and / or Microvascular embolism.

In another preferred example, the first preparation, the second preparation and the third preparation are administered simultaneously, separately or sequentially.

In another preferred example, the first preparation, the second preparation and the third preparation are administered before, during or after ischemia-reperfusion surgery, vascular stent surgery or thrombolytic surgery.

In another preferred example, the weight ratio of the first active ingredient, the second active ingredient, and the third active ingredient is 0.5-8: 0.5-6: 0.6-12, preferably 1-6: 0.5-4: 1- 10, more preferably 2-4: 1-3: 3-7.

According to a third aspect of the present invention, an active ingredient combination is provided. The active ingredient combination includes the following components:

(1) a therapeutically effective amount of a first active ingredient, the first active ingredient being clopidogrel or a pharmaceutically acceptable salt thereof;

(2) a therapeutically effective amount of a second active ingredient, said second active ingredient being salvianolic acid B or a pharmaceutically acceptable salt thereof; and

(3) A therapeutically effective amount of a third active ingredient, the third active ingredient being ginsenoside Rg1 or a pharmaceutically acceptable salt thereof.

In another preferred example, in the active ingredient combination, at least one active ingredient is independent.

According to a fourth aspect of the present invention, there is provided a composition according to the first aspect of the present invention, or a kit according to the second aspect of the present invention or an active ingredient combination according to the third aspect of the present invention for use in including : (I) Preparing a drug to improve bleeding and reducing microvascular obstruction during vascular stent surgery; (ii) Preparing a drug to improve bleeding and reducing microvascular obstruction during thrombolytic surgery; and / or (iii) Preparing to prevent and / or treat ischemia Reperfusion injury drugs.

In another preferred example, the ischemia-reperfusion injury includes bleeding injury and microvascular occlusion injury.

In another preferred example, the vascular stent operation is a vascular stent operation combined with anticoagulant drugs.

In another preferred example, the thrombolytic operation is a thrombolytic operation combined with an anticoagulant drug.

In another preferred example, the ischemia-reperfusion injury is selected from the group consisting of cardiovascular ischemia-reperfusion injury, cerebrovascular ischemia-reperfusion injury, out-of-circulation ischemia-reperfusion injury, and acute arterial embolization-reperfusion injury. , Reperfusion injury caused by traumatic shock reperfusion injury, surgical reperfusion injury, organ transplant reperfusion injury, burns, or impaired blood circulation caused by frostbite or thrombosis.

In another preferred example, the cardiovascular ischemia-reperfusion injury includes myocardial infarction, myocardial fibrosis, myocardial ischemia, myocardial necrosis, myocardial hypertrophy, heart failure or myocardial microvascular obstruction.

According to a fifth aspect of the present invention, there is provided the use of a compound combination, which comprises (a) salvianolic acid B or a pharmaceutically acceptable salt thereof and (b) ginsenoside Rg1 or a pharmaceutically acceptable salt thereof The compound combination is used to prepare a pharmaceutical composition or formulation, and the pharmaceutical composition or formulation is used to include: (i) improving bleeding and reducing microvascular obstruction during vascular stent surgery; (ii) improving thrombolytic surgery And reduce microvascular obstruction; and / or (iii) reduce the side effects of anticoagulants.

In another preferred example, the side effect of the anticoagulant drug is the use of an anticoagulant drug in a vascular stent operation or a thrombolytic operation, and the side effect produced by the anticoagulant drug.

In another preferred example, the side effects of the anticoagulant include bleeding and / or microcirculation disorders.

In another preferred example, the anticoagulant is selected from the group consisting of a vitamin K antagonist, an antiplatelet agglutination drug, a thrombin IIa inhibitor, a factor Xa inhibitor, or a combination thereof.

In another preferred example, the vitamin K antagonist is selected from the group consisting of warfarin.

In another preferred example, the anti-platelet agglutination drug is selected from the group consisting of aspirin, ticlopidine, clopidogrel, abciximab, etifebide or tirofiban, or a combination thereof.

In another preferred example, the thrombin IIa inhibitor is selected from the group consisting of bivalirudin, agatroban, dabigatran etexilate, and hirudin.

In another preferred example, the factor Xa inhibitor is selected from the group consisting of apixaban, rivaroxaban, edoxaban, or a combination thereof.

In another preferred example, the weight ratio of salvianolic acid B or a pharmaceutically acceptable salt thereof to ginsenoside Rg1 or a pharmaceutically acceptable salt thereof is 0.5-6: 0.6-12, preferably 0.5- 4: 1-10, more preferably 1-3: 3-7.

According to a sixth aspect of the present invention, there is provided a method for simultaneously reducing the risk of bleeding and microvascular obstruction, the method comprising the step of administering to a desired subject a composition according to the first aspect of the invention or a second aspect of the invention The kit.

In another preferred example, the subject is a patient undergoing surgery selected from the group consisting of ischemia-reperfusion surgery, vascular stent surgery, or thrombolytic surgery.

In another preferred example, the subject is a human and a non-human mammal.

In another preferred example, the object is a person.

In another preferred example, the non-human mammal includes (but is not limited to): pets (such as dogs and cats), domestic animals (such as cows, sheep, horses, and pigs), and various zoo animals (pandas, animals, and animals) Like) and so on.

It should be understood that, within the scope of the present invention, the above technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them here.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a hemodynamic detection in Example 1 of the present invention.

FIG. 2 is a Prussian blue staining diagram in Example 2 of the present invention.

FIG. 3 is a quantitative histogram of the area of cardiac hemorrhage in each group of rats in Example 2 of the present invention.

FIG. 4 is the result of Sirius Red staining in Example 3 of the present invention.

FIG. 5 is a result of hematoxylin and eosin staining in Example 4 of the present invention.

FIG. 6 is a Castales staining diagram in Example 5 of the present invention.

FIG. 7 is a histogram of quantitative results of cardiac microvascular obstruction in each group of rats in Example 5 of the present invention.

FIG. 8 shows Mallory phosphotungstate hematoxylin (PTAH) staining in the example of the present invention.

detailed description

After extensive and intensive research, the inventors have unexpectedly discovered that the combination of anticoagulant drugs (such as clopidogrel), salvianolic acid B, and ginsenoside Rg1, in ischemia-reperfusion injury, and in vascular stent surgery and Thrombolytic surgery can effectively reduce microvascular obstruction and effectively prevent bleeding. The combined therapeutic effect of the combination is obviously better than the three alone, and it can reduce the dosage of a single drug and reduce the toxicity of the drug. Based on this, the present invention has been completed.

Anticoagulant drugs are often used in ischemia-reperfusion, vascular stent surgery, and thrombolytic surgery. However, the situation of different patients (such as age, weight, condition, etc.) is often different, so this leads to anticoagulant drugs Difficulty of use. When the amount of anticoagulant drugs is too low, it often leads to insufficient anticoagulant effect, which leads to phenomena such as microvascular obstruction (or embolism); and when the amount of anticoagulant drugs is too high, it often leads to the risk of bleeding rise.

Unexpectedly, the present invention provides a dual or two-way synergistic protection effect, which can effectively reduce microvascular obstruction and effectively prevent bleeding, thereby greatly improving the efficacy of anticoagulant drugs and reducing their side effects, while significantly reducing Surgical risks of ischemia-reperfusion, vascular stent surgery, and thrombolytic surgery (including microvascular occlusion risk and bleeding (especially risk of hemorrhage and necrosis).

Specifically, the animal experiments of the present invention show that although "Clopidogrel + Salvianolic Acid B" can cause an increase in the risk of microvascular obstruction, "Clopidogrel + Ginsenoside Rg1" can also cause an increase in the risk of microvascular obstruction, but it is unexpected Yes, "Clopidogrel + Salvianolic Acid B / Ginsenoside Rg1" actually significantly reduced the risk of microvascular obstruction (and better than "Salvianolic Acid B + Ginsenoside Rg1").

Similarly, animal experiments have shown that although "Clopidogrel + Salvianolic Acid B" can increase the risk of bleeding, and "Clopidogrel + Ginsenoside Rg1" can also increase the risk of bleeding, but unexpectedly, "Clopidogrel "Gray + salvianolic acid B / ginsenoside Rg1" actually significantly reduced the risk of bleeding (and better than "salvianolic acid B + ginsenoside Rg1").

the term

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the terms "comprising," "including," and "containing" are used interchangeably and include not only closed definitions, but also semi-closed, and open definitions. In other words, the term includes "consisting of", "consisting essentially of".

As used herein, the ingredients of the term "pharmaceutically acceptable carrier" refer to those that are suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergies), that is, have a reasonable benefit / risk ratio substance.

As used herein, the term "therapeutically effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. Those of ordinary skill in the art should understand that the "therapeutically effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the excipients of the drugs used, the severity of the disease, and the combination with other drugs. A little different.

The "prevention" and "treatment" according to the present invention include delaying and stopping the progression of the disease, or eliminating the disease, and do not require 100% inhibition, elimination and reversal. In some embodiments, the composition or pharmaceutical composition of the present invention prevents, reduces, inhibits and / or reduces ischemia-reperfusion injury compared to the levels observed in the absence of the composition or pharmaceutical composition of the present invention. Or reversed, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%.

Active ingredient

In the present invention, the first active ingredient is clopidogrel or a pharmaceutically acceptable salt thereof, the second active ingredient is salvianolic acid B or a pharmaceutically acceptable salt thereof, and the third active ingredient is ginsenoside Rg1.

Clopidogrel is an anticoagulant and antiplatelet drug used to prevent and treat heart, brain and other arterial circulation disorders caused by high platelet aggregation, such as recent stroke, myocardial infarction and diagnosed peripheral arteries disease.

Salvianolic acid B has a strong antioxidant effect and is one of the natural products with the strongest antioxidant effect known at present; it significantly improves the neurological deficits of rats with cerebral ischemia reperfusion injury, which is manifested as an improvement in behavioral disorders. Reduce the area of cerebral infarction; delay the protection of cardiac microvascular endothelial cells; prevent and cure atherosclerosis; protect the pre-adapted heart cells.

Ginsenoside Rg1 has the effects of slowing heart rate and two-way blood pressure regulation; relaxing blood vessels, improving neural plasticity, enhancing learning and memory, anti-aging, anti-fatigue, improving immunity, assisting anti-tumor, repairing functions and so on.

As used herein, "clopidogrel" refers to clopidogrel, or a pharmaceutically acceptable salt thereof. It should be understood that the term also includes mixtures of the aforementioned components.

As used herein, "salfinic acid B" refers to salvianolic acid B, or a pharmaceutically acceptable salt thereof. It should be understood that the term also includes mixtures of the aforementioned components.

As used herein, "ginsenoside Rg1" refers to ginsenoside Rg1, or a pharmaceutically acceptable salt thereof. It should be understood that the term also includes mixtures of the aforementioned components.

In the present invention, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention and an acid or base suitable for use as a medicament. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred type of salt is a salt of a compound of the invention with an acid. Suitable acids for forming salts include, but are not limited to, inorganic acids such as toluenesulfonic acid, hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, Organic acids such as fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid. A preferred type of salt is a salt formed by a compound of the present invention with a base. Suitable bases for forming the salt include, but are not limited to, inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and sodium phosphate, ammonia, and Organic bases such as ethylamine and diethylamine. A preferred type of salt is a salt formed by a compound and a metal ion. Metal ions suitable for forming a salt include, but are not limited to, sodium ion, magnesium ion, potassium ion, calcium ion, and the like.

A typical pharmaceutically acceptable salt of clopidogrel is clopidogrel sulfate, clopidogrel hydrogen sulfate, or clopidogrel hydrochloride.

A typical pharmaceutically acceptable salt of salvianolic acid B is salvianolic acid B magnesium salt, salvianolic acid B calcium salt.

Representatively, the chemical structural formulas of clopidogrel sulfate, salvianolic acid B, and ginsenoside Rg1 are as follows:

Composition, kit, active ingredient combination and pharmaceutical composition

The invention provides a composition comprising:

In another preferred example, the weight ratio of clopidogrel or a pharmaceutically acceptable salt thereof, salvianolic acid B or a pharmaceutically acceptable salt thereof, and ginsenoside Rg1 is 0.5-8: 0.5-6: 0.6-12 , Preferably 1-6: 0.5-4: 1-10, more preferably 2-4: 1-3: 3-7.

In another preferred example, the purity of the ginsenoside Rg1 is 80-100%, preferably 90% -100%.

If necessary, the composition may further include a pharmaceutically acceptable carrier to prepare a pharmaceutical composition (drug).

Typically, the composition further includes a pharmaceutically acceptable carrier to make a pharmaceutical composition, and the pharmaceutical composition includes:

(b) a therapeutically effective amount of a second active ingredient, said second active ingredient being salvianolic acid B or a pharmaceutically acceptable salt thereof;

(c) a therapeutically effective amount of a third active ingredient, said third active ingredient being ginsenoside Rg1 or a pharmaceutically acceptable salt thereof; and

(d) a pharmaceutically acceptable pharmaceutical carrier.

The pharmaceutical composition containing the first active ingredient, the second active ingredient, and the third active ingredient according to the present invention may be various dosage forms suitable for oral administration, and may also be various external administration preparations or other gastrointestinal Preparations for external administration. For example, the preparation for external administration according to the present invention may be further supplemented with auxiliary materials such as a surfactant, a transdermal absorption enhancer, a preservative, a solvent, an antioxidant, a humectant, a pH adjuster, a colorant, and a fragrance. Prepared (including but not limited to): tinctures, tinctures, oils, ointments, plasters, pastes, ironing agents, plasters, patches, film coatings, films, gels, rags, Acupoint patches, sprays, aerosols, implants, emulsions, etc. For ischemia-reperfusion injury, the preferred dosage forms include: various dosage forms for oral administration, implants, and injections.

It should be understood that, in the present invention, the carrier is not particularly limited, and is a material commonly used in the art, and its kind, method of use, and source are well known to those skilled in the art.

Examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, etc.), gelatin, talc, and solid lubricants. (Such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium lauryl sulfate), buffering agents, chelating agents, thickeners, pH adjusters, transdermal enhancers, colorants, flavoring agents, stabilizers, antioxidants, preservatives , Bacteriostatic agent, pyrogen-free water, etc.

In another preferred example, in the pharmaceutical composition, the content range of the first active ingredient and the second active ingredient, and the quality of the first active ingredient and the second active ingredient are as described in the above composition. Description.

The present invention also provides an active ingredient composition. The active ingredient combination includes the following components: (1) a therapeutically effective amount of a first active ingredient, wherein the first active ingredient is clopidogrel or a pharmaceutically acceptable Salt

In the active ingredient combination, the first active ingredient, the second active ingredient, and the third active ingredient may be independent of each other, or may be combined together to exist in the form of an active ingredient composition. Preferably, in the active ingredient combination, at least one active ingredient is independent.

The invention also provides a pill box, which comprises:

(D) Instruction manual.

In another preferred example, the instructions for use indicate that the first preparation, the second preparation and the third preparation are used in combination to prevent and / or treat ischemia-reperfusion, ischemia and / or microvascular Blocking injury.

In another preferred example, the first preparation, the second preparation and the third preparation are administered simultaneously, separately or sequentially in the prevention and / or treatment of ischemia-reperfusion injury, ischemia and / or microvascular obstruction. Dosing.

In the kit according to the present invention, it should be understood that the first formulation, the second formulation and the third formulation in the kit may be independent of each other, or any two or three formulations may be combined. of.

For example, the second and third formulations are combined and the first formulation is independent. Typically, the kit of the present invention comprises: one tablet (first preparation) and one injection (combined preparation of second preparation and third preparation), wherein the tablet contains clopidogrel or a pharmaceutically acceptable The salt contains salvianolic acid B or a pharmaceutically acceptable salt thereof and ginsenoside Rg1 or a pharmaceutically acceptable salt thereof.

In another preferred example, in the kit, the clopidogrel or a pharmaceutically acceptable salt thereof, salvianolic acid B or a pharmaceutically acceptable salt thereof, and ginsenoside Rg1 or a pharmaceutically acceptable salt thereof An acceptable weight ratio of salt is 0.5-8: 0.5-6: 0.6-12, preferably 1-6: 0.5-4: 1-10, and more preferably 2-4: 1-3: 3-7.

The composition, active ingredient combination, pharmaceutical composition, and kit of the present invention can be prepared by conventional methods and equipment.

Uses and administration

The invention provides a composition, an active ingredient combination, a pharmaceutical composition, or a kit as described herein.

For including: (i) preparing a drug to improve bleeding and reducing microvascular obstruction during vascular stent surgery; (ii) preparing a drug to improve bleeding and reducing microvascular obstruction during thrombolytic surgery; and / or (iii) preparing to prevent and / or Drugs for the treatment of ischemia-reperfusion injury.

Preferably, the ischemia-reperfusion injury includes (but is not limited to): cardio-cerebrovascular ischemia-reperfusion injury, out-of-circulation ischemia-reperfusion injury, acute arterial embolism reperfusion injury, traumatic shock reperfusion injury, surgery Reperfusion injury due to surgical reperfusion injury, organ transplantation reperfusion injury, burn, frostbite, or blood circulation disorders due to thrombosis.

In another preferred example, the cardiovascular ischemia-reperfusion injury includes myocardial infarction, myocardial fibrosis, myocardial ischemia, myocardial necrosis, myocardial hypertrophy, heart failure or myocardial microvascular obstruction

The composition, the active ingredient combination, the pharmaceutical composition and the first active ingredient, the second active ingredient, and the third active ingredient in the kit can prevent and treat ischemia-reperfusion injury, bleeding, and microvascular obstruction. Produces synergistic effects, enhances the effect of treating ischemia-reperfusion injury, reduces the dosage of a single drug, and reduces drug toxicity.

Before, simultaneously or after using the composition, active ingredient combination, pharmaceutical composition and kit of the present invention, it can be used in combination with other active substances for treating ischemia-reperfusion injury or combined with surgery for ischemia-reperfusion injury. .

In the process of combined use, drug interactions are divided into additive effects, synergistic effects, and antagonistic effects according to the effects of drugs in common use. Synergistic effects mean that the effects of combined drugs are significantly stronger than those of single drugs. Harmony means that the effect of the combined drug is equivalent to that of the drug used alone, and antagonism means that the effect of the combined drug is smaller than that of the drug used alone. In the present invention, it is found for the first time that the first active ingredient, the second active ingredient, and the third active ingredient are used in combination to have a synergistic effect.

When preventing and / or treating ischemia-reperfusion injury, the administration method of the present invention includes sequentially applying the first active ingredient, the second active ingredient, and the third active ingredient, or the first active ingredient and the second active ingredient simultaneously. And a third active ingredient.

The pharmaceutical preparation should match the mode of administration. When using a pharmaceutical composition or preparation, a safe and effective amount of the drug is administered to a desired subject (such as a human or non-human mammal). The dosage used is usually at least about 0.1 mg, and in most cases no more than about 2500 mg. Preferably, the dose is from 1 mg to 500 mg; the safe and effective amount of the second active ingredient is usually at least about 0.01 mg, and in most cases no more than 2500 mg. Preferably, the dosage range is from 0.1 mg to 2500 mg. The safe and effective amount of the third active ingredient is usually at least about 0.01 mg, and in most cases it does not exceed 2500 mg. Preferably, the dosage range is from 0.1 mg to 2500 mg. Of course, the specific dose should also consider the route of administration, the patient's health and other factors, which are all within the skill of a skilled physician. When the first active ingredient, the second active ingredient, and the third active ingredient are applied successively, there is no particular requirement for the interval of application. The composition, the active ingredient combination, the pharmaceutical composition and the first active ingredient, the second active ingredient, and the third active ingredient in the kit of the present invention are administered simultaneously or sequentially in the same or different routes, respectively, including but not Limited to: oral administration, injection administration, intratumoral administration, implantation administration, intracavity administration, anal administration, transdermal administration, internal and external application;

Preferred injections include: intravenous, intramuscular, subcutaneous, and intracavitary injection.

The present invention also provides the use of a combination of compounds, the compound combination comprising salvianolic acid B or a pharmaceutically acceptable salt thereof and ginsenoside Rg1 or a pharmaceutically acceptable salt thereof, and the compound combination is used for preparing A pharmaceutical composition or formulation for use in: (i) improving bleeding and reducing microvascular obstruction during vascular stent surgery; (ii) improving bleeding and reducing microvascular obstruction during thrombolytic surgery; and / Or (iii) reduce the side effects of anticoagulants.

In the present invention, the combination of salvianolic acid B and ginsenoside Rg1 can improve the side effects of anticoagulant drugs. The anticoagulant drugs are not particularly limited. Representatively, the anticoagulant drugs include (but are not limited to) : Vitamin K antagonist, antiplatelet agglutination drug, thrombin IIa inhibitor, factor Xa inhibitor, or a combination thereof.

Typically, the vitamin K antagonist includes (but is not limited to): warfarin.

Typically, the anti-platelet agglutination drug includes (but is not limited to): aspirin, ticlopidine, clopidogrel, abciximab, etifebide or tirofiban, or a combination thereof.

Typically, the thrombin IIa inhibitor includes (but is not limited to): bivalirudin, agatroban, dabigatran etexilate, hirudin.

Typically, the factor Xa inhibitor includes (but is not limited to): apixaban, rivaroxaban, edoxaban, or a combination thereof

Preferably, the weight ratio of salvianolic acid B or a pharmaceutically acceptable salt thereof to ginsenoside Rg1 or a pharmaceutically acceptable salt thereof is 0.5-6: 0.6-12, preferably 0.5-4: 1 -10, more preferably 1-3: 3-7.

The invention also provides a method for simultaneously reducing the risk of bleeding and microvascular obstruction, said method comprising the step of administering to a desired subject a composition, an active ingredient combination, a pharmaceutical composition or a medicament according to the invention box.

In another preferred example, the subject is a human and a non-human mammal. Typically, the non-human mammal includes (but is not limited to): pets (such as dogs, cats), domestic animals (such as cattle, sheep, horses, pigs), various zoo animals (panda, elephant, tiger, etc.) )Wait.

The main advantages of the invention include:

1. In the present invention, the combination of clopidogrel, salvianolic acid B and ginsenoside Rg1 significantly improves the clinical efficacy of ischemia-reperfusion, ischemia and / or microvascular obstruction, and significantly improves the structure and organ structure of ischemia-reperfusion injury. The adverse effects of the function significantly improve the risk of intracardiac hemorrhage caused by thrombolytic drugs or anticoagulant drugs and the induced heart failure and cardiovascular obstruction during PCI (coronary stent surgery) or thrombolytic therapy.

2. In the present invention, the combined use of salvianolic acid B and ginsenoside Rg1 can obviously improve side effects such as bleeding or microcirculation disturbance of anticoagulant drugs (such as clopidogrel).

The present invention will be further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally based on conventional conditions or conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

Example 1

Clopidogrel combined with salvianolic acid B / ginsenoside Rg1 improves cardiac function in rats with ischemia-reperfusion

1.1 Experimental materials

1.1.1 Experimental animals

Wistar rats: 90 males, provided by Shanghai Experimental Animal Center, Chinese Academy of Sciences, kept in SPF animal room of Shanghai Animal Research Center of Chinese Academy of Sciences, constant temperature 22 ± 2 ℃, 12h light, standard diet, free drinking water.

1.1.2 Drugs

Salvianolic acid B and ginsenoside Rg1 were purchased from Shanghai Yousi Biological Technology Co., Ltd. (purity ≥99%). Clopidogrel was purchased from Sanofi (Hangzhou) Pharmaceutical Co., Ltd.

Drug configuration method: ① Weigh 1.5mg salvianolic acid B and dissolve it in 1ml physiological saline, filter with a microporous filter, and vortex for 3 minutes until the drug is completely dissolved. ② Weigh 1.5mg of ginsenoside Rg1 and dissolve it in 1ml of physiological saline, filter it with a microporous filter, and vortex for 3 minutes until the drug is completely dissolved. ③ Weigh out 0.43mg salvianolic acid B and 1.07mg ginsenoside Rg1 and mix well (1.5mg in total) to dissolve in 1ml physiological saline, filter through a microporous membrane, and vortex for 3 minutes until the mixture is completely dissolved to obtain 2: 5 Pharmaceutical composition. ④ Weigh 0.675 mg of clopidogrel and dissolve it in 1 ml of physiological saline, filter it with a microporous filter, and vortex for 3 minutes until the drug is completely dissolved.

1.2 Experimental methods

1.2.1 Preparation of myocardial ischemia-reperfusion injury model

After weighing the rats, the clopidogrel group and the combination group with clopidogrel were intragastrically administered with 6.75 mg / kg clopidogrel and intraperitoneally injected with a mixed anesthetic agent of 60 mg / kg ketamine and 6 mg / kg xylazine hydrochloride. . The rats were fixed on the operation board by the back fixation method, the chest was covered with hair, and the skin was disinfected by rubbing iodine. The intravenous indwelling needle sleeve was inserted into the trachea and connected to a ventilator. The breathing frequency was set to 80 times / minute, the breathing ratio was 1: 1, and the tidal volume was 18 ml. Cut the skin longitudinally about 1 cm along the left edge of the sternum, bluntly separate the muscles, and open the gap between the third and fourth ribs and fix it. Hemostatic forceps clamp the thymus and pull upwards to expose the upper part of the heart. Find the anterior descending coronary artery between the pulmonary artery cone and the left atrial appendage. Use a non-invasive suture needle 5-0 silk thread to pass through the anterior descending coronary artery 2mm below the beginning of the coronary artery. Knots everywhere, myocardial tissue was pale after successful ligation of the left anterior descending branch. After 40 minutes, the non-invasive suture was cut to restore ischemic myocardium to perfusion. After reperfusion, the muscles and skin were sutured, and the tail vein was administered at the same time. The rats were kept in a 37-degree constant temperature system to keep warm, and then returned to their cages after waking up. The sham operation is exactly the same except that the left anterior descending coronary artery is not ligated. Hemodynamic evaluation was performed 7 days after reperfusion.

1.2.2 Animal grouping and administration

About 220g rats were randomly divided into the following 9 groups: sham operation group, ischemia-reperfusion model group, salvianolic acid B group (administration amount: 15mg / kg), ginsenoside Rg1 group (administration amount: 15mg / kg) 1. Salvianolic acid B / ginsenoside Rg1 combined group (wherein, the dose of salvianolic acid B is 4.29 mg / kg, ginsenoside Rg1 dose is 10.71 mg / kg), clopidogrel group (dosage amount 6.75 mg / kg), a combination of clopidogrel + salvianolic acid B (wherein the dose of clopidogrel is 6.75 mg / kg and the dose of salvianolic acid B is 15 mg / kg), clopidogrel + Ginsenoside Rg1 combined group (wherein the dose of clopidogrel was 6.75 mg / kg, ginsenoside Rg1 dose was 15 mg / kg), clopidogrel + salvianolic acid B / ginsenoside Rg1 combined group ( Among them, the dosage of clopidogrel was 6.75 mg / kg, the dosage of salvianolic acid B was 4.29 mg / kg, and the dosage of ginsenoside Rg1 was 10.71 mg / kg), with 10 mice in each group.

1.2.3 Hemodynamic detection

After intraperitoneal injection of 60 mg / kg ketamine and 6 mg / kg xylazine hydrochloride mixed anesthesia in rats, the right common carotid artery was isolated and a Miller catheter was inserted. The carotid artery pressure and left were recorded with a Powerlab 8/30 physiological recorder (ML870, ADINSTRUMENTS). Hemodynamic indexes such as ventricular pressure, maximum left ventricular systolic velocity, maximum left ventricular diastolic velocity, left ventricular end-systolic pressure, and left ventricular systolic pressure.

1.2.4 Statistical methods

All experimental data are expressed as mean ± standard deviation (Mean ± SD). SPSS 19.0 software was used to perform statistical analysis on the data. The comparison between multiple groups was based on a completely random design one-way analysis of variance (one-way ANVOVA). The pairwise comparison between multiple sample means was performed using the Bonferroni method; P <0.05 was considered statistically significant学 significance.

1.3 Experimental results

The hemodynamic test results of different experimental groups are shown in Table 1 and Figure 1.

Table 1 Hemodynamic testing results

Note: * p <0.05, ** p <0.01, *** p <0.001VS sham operation group, #p <0.05, ## p <0.01, ### p <0.001VS model group, △ p <0.05, △△ p <0.01, △△△ p <0.001VS clopidogrel group, n≥8.

As can be seen from Table 1 and Figure 1, compared with the sham operation control group, the maximum left ventricular systolic rate, maximum left ventricular diastolic rate, and systolic blood pressure in the ischemia-reperfusion model group were significantly reduced, and end-diastolic blood pressure was significantly increased. Compared with the ischemia-reperfusion model group, the salvianolic acid B / ginsenoside Rg1 group significantly increased the maximum contraction rate; compared with the ischemia-reperfusion model, the combination of clopidogrel + ginsenoside Rg1 significantly increased the left ventricular maximum contraction rate. Compared with clopidogrel alone, the maximum left ventricular contraction rate was significantly increased. Compared with clopidogrel + salvianolic acid B / ginsenoside Rg1 and ischemia-reperfusion model, the left ventricular maximum contraction rate, left ventricular maximum diastolic rate and systolic blood pressure were significantly increased, and clopidogrel + salvianolic acid B Compared with clopidogrel alone, the maximum systolic rate and systolic blood pressure of left ventricle were significantly increased in combination with ginsenoside Rg1.

Example 2

Clopidogrel combined with salvianolic acid B / ginsenoside Rg1 improves myocardial hemorrhage in ischemia-reperfusion rats

2.1 Experimental materials

2.1.1 Experimental animals

The experimental animals are the same as 1.1.1 in Example 1.

2.2 Experimental methods

2.2.1 Preparation of myocardial ischemia-reperfusion model

The method was the same as that in 1.2.1.

2.2.2 Animal grouping and administration

The method was the same as that in Example 1.2.2.

2.2.3 Prussian blue staining

Paraffin section thickness is 4 μm. First, paraffin tissue is baked (65 ° C, 45-50 minutes), and then dewaxed to the aqueous phase (xylene 15 minutes, absolute ethanol 5 minutes, 95% ethanol 5 minutes, 75% Ethanol for 3 minutes, rinse with distilled water for 1 minute); Prussian blue staining solution was dripped on the sections for 45 minutes, and then washed with distilled water for 2 minutes; the sections were placed in eosin staining solution for 10 minutes, and tap water was washed for 3 seconds; After dehydration, permeabilize with xylene (75% ethanol for 2 minutes, 95% ethanol for 2 minutes, absolute ethanol for 3 minutes, and xylene for 15 minutes), and seal with neutral gum.

2.2.4 Quantification of Prussian Blue Staining

After the Prussian blue staining is completed, the blue dots represent bleeding. Each sample of Prussian blue staining is photographed at the same magnification, and the bleeding area and left ventricle area are quantified, and the percentage of bleeding area to the left ventricle is calculated.

2.3 Experimental results

The Prussian blue staining results are shown in Figure 2. It can be seen that the left ventricular hemorrhage is obvious in the ischemia-reperfusion model group compared with the sham-operated control group; the clopidogrel + salvianolic acid B / ginsenoside Rg1 combined with ischemia-reperfusion The perfusion injury model group has significantly improved bleeding compared with the clopidogrel + salvianolic acid B / ginsenoside Rg1 combination group, which has more significant bleeding improvement effects than the salvianolic acid B / ginsenoside Rg1 group; and the salvianolic acid B group alone The ginsenoside Rg1 group, the clopidogrel group alone, the clopidogrel + salvianolic acid B group, and the clopidogrel + ginsenoside Rg1 group had no significant improvement in bleeding compared with the ischemia-reperfusion model group.

Prussian blue staining shows the quantitative results of heart bleeding area of each group of rats as shown in Table 2 and Figure 3:

Table 2 Quantification of heart hemorrhage area in each group of rats

Note: * p <0.05, ** p <0.01, *** p <0.001VS sham operation group, #p <0.05, ## p <0.01, ### p <0.001VS model group, n≥6.

It can be seen from Table 2 that the left ventricular hemorrhage of the ischemia-reperfusion injury model group is significantly increased compared with the sham operation control group. The clopidogrel group alone has a certain bleeding risk but is improved than the model group. The combination of clopidogrel with salvianolic acid B and the combination of clopidogrel with ginsenoside Rg1 increased the bleeding area compared with clopidogrel alone, increasing the risk of bleeding, and no improvement was found; however, it was unexpectedly found that clopidogrel and Compared with salvianolic acid B / ginsenoside Rg1 compared with clopidogrel alone, it can significantly reduce the bleeding area and significantly reduce the risk of bleeding. Therefore, it can be concluded that salvianolic acid B / ginsenoside Rg1 can significantly reduce the risk of bleeding.

Example 3

Clopidogrel combined with salvianolic acid B / ginsenoside Rg1 improves myocardial fibrosis in ischemia-reperfusion rats

3.1 Experimental materials

3.1.1 Experimental animals

The experimental animals are the same as 1.1.1 in Example 1.

3.2 Experimental methods

3.2.1 Preparation of myocardial ischemia reperfusion model

The method was the same as that in 1.2.1.

3.2.2 Animal grouping and administration

The method was the same as that in Example 1.2.2.

3.2.3 Sirius Red Stain

The paraffin slices were baked at 65 ° C for 45-50 minutes, then placed in xylene for 15 minutes, absolute ethanol for 5 minutes, 95% ethanol for 5 minutes, 75% ethanol for 3 minutes, and running water for 1 minute to dewax; Sirius was dyed red The droplets were added to the sections for 1 hour, and then rinsed with running water for 2 minutes; dehydrated and permeabilized with xylene (1 minute of absolute ethanol and 10 minutes of xylene), and sealed with neutral gum.

3.3 Experimental results

Sirius red staining results are shown in Figure 4. Compared with the sham operation control group, the left ventricle of the ischemia-reperfusion model group has a large amount of collagen deposition leading to myocardial fibrosis; the clopidogrel + salvianolic acid B / ginsenoside Rg1 combination group And the salvianolic acid B / ginsenoside Rg1 group has significantly reduced collagen deposition and myocardial fibrosis compared with the ischemia-reperfusion model group, and the clopidogrel + salvianolic acid B / ginsenoside Rg1 group is better than salvianol. Acid B / ginsenoside Rg1 group more significantly reduced collagen deposition and improved fibrosis; while salvianolic acid B group alone, ginsenoside Rg1 group alone, clopidogrel group alone, clopidogrel + salvianolic acid group B and clopidogrel Compared with the ischemia-reperfusion model group, the gray + ginsenoside Rg1 group had no significant reduction in collagen and no significant improvement in fibrosis.

Example 4

Clopidogrel combined with salvianolic acid B / ginsenoside Rg1 improves myocardial structure in rats with ischemia-reperfusion

4.1 Experimental materials

4.1.1 Experimental animals

The experimental animals are the same as 1.1.1 in Example 1.

4.2 Experimental methods

4.2.1 Preparation of myocardial ischemia-reperfusion model

The method was the same as that in 1.2.1.

4.2.2 Animal grouping and administration

The method was the same as that in Example 1.2.2.

4.2.3 Hematoxylin-eosin staining

The paraffin tissue is baked (65 ° C, 45-50 minutes), and then dewaxed to the water phase (xylene 15 minutes, absolute ethanol 5 minutes, 95% ethanol 5 minutes, 75% ethanol 3 minutes, and running water rinse 1 Minutes); put into hematoxylin staining solution for 15 minutes, then rinse with running water for 4 minutes; differentiate with 1% hydrochloric acid ethanol for 10 seconds (differentiation time is adjusted according to the time when the differentiation solution is placed), and rinse with water flowing for 5 minutes ; Eosin staining solution stained for 1 minute and placed in water for 1 minute; dehydrated and permeabilized with xylene (75% ethanol for 2 minutes, 95% ethanol for 4 minutes, anhydrous ethanol for 4 minutes, and xylene for 15 minutes), neutral gum Cover film.

4.3 Experimental results

The hematoxylin and eosin staining results are shown in FIG. 5. Compared with the sham operation control group, the ischemia-reperfusion model group has irregular cell arrangement, necrosis and a large number of inflammatory cell infiltration; clopidogrel + salvianolic acid B / ginsenoside Rg1 combined group and salvianolic acid B / ginseng Compared with the ischemia-reperfusion model group, the cell arrangement of the saponin Rg1 group is relatively regular, the number of necrotic cells is reduced, and the inflammation infiltration is significantly improved. The clopidogrel + salvianoside Rg1 combined group and salvianoside B / Compared with the ginsenoside Rg1 group, the cell arrangement of the clopidogrel + salvianolic acid B / ginsenoside Rg1 combination group was more regular, the necrosis was reduced, and the inflammation infiltration was improved; while the salvianolic acid B group alone and the ginsenoside Rg1 group Compared with the ischemia-reperfusion model group, the cell arrangement, necrosis and inflammatory infiltration showed a certain degree of improvement in the clopidogrel group alone, the clopidogrel + salvianolic acid B group and the clopidogrel + ginsenoside Rg1 group.

Example 5

Clopidogrel combined with salvianolic acid B / ginsenoside Rg1 improves myocardial microvascular disorder in rats with ischemia-reperfusion

5.1 Experimental materials

5.1.1 Experimental animals

The experimental animals are the same as 1.1.1 in Example 1.

5.2 Experimental methods

5.2.1 Preparation of myocardial ischemia-reperfusion model

The method was the same as that in 1.2.1.

5.2.2 Animal grouping and administration

The method was the same as that in Example 1.2.2.

5.2.3 Castales Staining

The paraffin tissue is baked (65 ° C, 45-50 minutes), and then dewaxed to the water phase (xylene 15 minutes, absolute ethanol 5 minutes, 95% ethanol 5 minutes, 75% ethanol 3 minutes, and running water rinse 1 Min); add GENMED fixing solution on the section, and incubate for 72 hours at room temperature; remove the fixing solution and add cleaning solution and incubate for 2 minutes; remove the cleaning solution and add GENMED mordant solution and incubate for 8 minutes at room temperature; remove GENMED mordant solution and add cleaning solution Incubate for 2 minutes; remove the cleaning solution and incubate for 5 minutes at room temperature; remove the GENMED solution and incubate for 5 minutes; remove the cleaning solution and incubate for 45 minutes with GENMED orange stain; remove the GENMED orange stain Add cleaning solution and incubate for 2 minutes; remove cleaning solution and add GENMED fuchsin solution and incubate for 2 minutes; remove GENMED fuchsin solution and add cleaning solution and incubate for 2 minutes; remove cleaning solution and add GENMED acid solution to incubate for 2 minutes; remove GENMED acid Add cleaning solution and incubate for 2 minutes; remove cleaning solution and add GENMED staining solution and incubate for 15 minutes; remove GENMED staining solution and add cleaning solution and incubate for 2 minutes; dehydrate and then permeabilize with xylene (1 minute of absolute ethanol, xylene 15 Bell), mounted with neutral gum.

5.2.3 Quantitative Methods of Castales Staining

Use the BX51 microscope to take pictures of the infarcted area of each sample one by one, and try to achieve a full coverage of the infarcted area. Because the infarct area of each group of animals is different, the number of photos taken for each heart sample ranges from 10-30. In order to ensure that the infarct lesion area of each sample is covered and shot, the number of microvascular obstructions in each film is collected and counted. The aggregated red blood cells, platelets, or leukocytes are greater than or equal to 4 and are defined as microvascular obstructions. Based on the number of infarcted blood vessels taken by the animals, statistics were obtained for each group of animals to obtain the average number of infarcted blood vessels and the standard deviation for each group of animals.

5.3 Experimental results

As shown in Figure 6 of the Castales stained photograph. Compared with the sham operation control group, there were a large number of microvascular obstructions in the ischemia-reperfusion injury model group, with red blood cells, platelets, and white blood cells clustering in the microvessels; the combination of clopidogrel + salvianolic acid B / ginsenoside Rg1 and ischemia-reperfusion Compared with the model group, the number of occluded microvessels was significantly reduced, and the microvascular disorder was significantly improved. The clopidogrel + salvianolic acid B / ginsenoside Rg1 combination group and the salvianolic acid B / ginsenoside Rg1 group had a more significant reduction in the number of microvascular infarctions. The effect of improving microvascular disorders is more obvious; while salvianolic acid B alone, ginsenoside Rg1 alone, clopidogrel alone, clopidogrel + salvianolic acid B and clopidogrel + ginsenoside Rg1, Compared with the perfusion model group, the microvascular obstruction was not significantly improved.

The quantitative results of the number of cardiac microvascular obstructions of rats in each group of Castales staining are shown in Table 3 and Figure 7:

Table 3 Quantification of cardiac microvascular obstruction in each group of rats

组别Group	微血管阻塞平均数(个)Microvascular obstruction (average)
假手术组mock surgical group	0.00±0.000.00 ± 0.00
缺血再灌注模型组Ischemia-reperfusion model group	5.64±1.28 ^ 5.64 ± 1.28 ^
氯吡格雷组Clopidogrel	4.51±0.664.51 ± 0.66
氯吡格雷+丹酚酸B组Clopidogrel + salvianolic acid group B	5.04±3.075.04 ± 3.07
氯吡格雷+人参皂苷Rg1组Clopidogrel + ginsenoside Rg1 group	7.57±6.267.57 ± 6.26
氯吡格雷+丹酚酸B/人参皂苷Rg1组Clopidogrel + salvianolic acid B / ginsenoside Rg1 group	2.26±1.58 ^### 2.26 ± 1.58 ^###

Table 3 can more objectively and comprehensively evaluate the improvement effect of drugs on microcirculation disorders. Table 3 quantitative results show that the number of blocked microvessels in the ischemia-reperfusion injury model group is significantly increased compared with the sham operation control group (P <0.01). Compared with the ischemia-reperfusion model group, the salvianolic acid B / ginsenoside Rg1 combination significantly reduced the number of occluded microvessels, and the microvascular disorders were significantly improved (p <0.001). Both clopidogrel and salvianolic acid B in combination with clopidogrel and ginsenoside Rg1 increased the number of microvascular obstructions and increased the risk of embolism compared with clopidogrel alone; however, it was unexpectedly found that clopidogrel and Compared with clopidogrel alone, salvianolic acid B / ginsenoside Rg1 can significantly reduce the number of microvascular obstructions, significantly reduce the risk of embolism, and significantly improve myocardial microvascular disorders. Therefore, salvianolic acid B / ginsenoside can be obtained Rg1 can significantly reduce the risk of microvascular obstruction.

Example 6

Clopidogrel combined with salvianolic acid B / ginsenoside Rg1 improves rhabdomyolysis in ischemia-reperfusion rats

6.1 Experimental materials

6.1.1 Experimental animals

The experimental animals are the same as 1.1.1 in Example 1.

6.2 Experimental methods

6.2.1 Preparation of myocardial ischemia-reperfusion model

The method was the same as that in 1.2.1.

6.2.2 Animal grouping and administration

The method was the same as that in Example 1.2.2.

6.2.3 Mallory Phosphotungstate Hematoxylin (PTAH) Staining

The paraffin tissue is baked (65 ° C, 45-50 minutes), and then dewaxed to the water phase (xylene 15 minutes, absolute ethanol 5 minutes, 95% ethanol 5 minutes, 75% ethanol 3 minutes, and distilled water rinse 2 Minutes); put in potassium permanganate solution for 10 minutes to oxidize; wash in tap water for 1 minute, distilled water for 1 minute, and bleach in oxalic acid solution for 2 minutes; Staining in hematoxylin solution for 48 hours; direct differentiation into 95% alcohol for 30 seconds, rapid dehydration of absolute ethanol, and transparency with xylene (30 seconds of absolute ethanol, 15 minutes of xylene), and sealing with neutral gum.

6.3 Experimental results

The striated muscle tissue Mallory phosphotungstate hematoxylin (PTAH) staining results are shown in Figure 8. Compared with the sham operation control group, the striated muscle tissue in the ischemia-reperfusion model group was largely dissolved and broken; the clopidogrel + salvianolic acid B / ginsenoside Rg1 combined group and the salvianolic acid B / ginsenoside Rg1 group were separately Compared with the striated muscle group, the perfusion model group had significantly reduced P dissolution, and the clopidogrel + salvianolic acid B / ginsenoside Rg1 combined group compared with the salvianolic acid B / ginsenoside Rg1 group, the clopidogrel + salvianoside B / ginseng Rhabdomyolysis was further reduced in the saponin Rg1 combination group; salvianolic acid B alone, ginsenoside Rg1 alone, clopidogrel alone, clopidogrel + salvianolic acid B and clopidogrel + ginsenoside Rg1 Compared with the blood reperfusion model group, rhabdomyolysis was not significantly improved.

All documents mentioned in the present invention are incorporated by reference in this application, as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

A composition, characterized in that the composition includes:

(a) a therapeutically effective amount of a first active ingredient, the first active ingredient being clopidogrel or a pharmaceutically acceptable salt thereof;

(b) a therapeutically effective amount of a second active ingredient, said second active ingredient is salvianolic acid B or a pharmaceutically acceptable salt thereof; and

(c) a therapeutically effective amount of a third active ingredient, the third active ingredient being ginsenoside Rg1 or a pharmaceutically acceptable salt thereof.
The composition according to claim 1, wherein the clopidogrel or a pharmaceutically acceptable salt thereof, salvianolic acid B or a pharmaceutically acceptable salt thereof, and ginsenoside Rg1 or a pharmaceutically acceptable compound thereof The weight ratio of the accepted salt is 0.5-8: 0.5-6: 0.6-12, preferably 1-6: 0.5-4: 1-10, more preferably 2-4: 1-3: 3-7.
The composition according to claim 1, wherein the composition is a pharmaceutical composition.
A pill box, characterized in that the pill box includes:

(A) a first formulation containing clopidogrel or a pharmaceutically acceptable salt thereof;

(B) a second formulation containing salvianolic acid B or a pharmaceutically acceptable salt thereof;

(C) a third formulation containing ginsenoside Rg1 or a pharmaceutically acceptable salt thereof; and

(D) Instruction manual.
An active ingredient combination, characterized in that the active ingredient combination includes the following components:

(1) a therapeutically effective amount of a first active ingredient, the first active ingredient being clopidogrel or a pharmaceutically acceptable salt thereof;

(2) a therapeutically effective amount of a second active ingredient, said second active ingredient being salvianolic acid B or a pharmaceutically acceptable salt thereof; and

(3) A therapeutically effective amount of a third active ingredient, the third active ingredient being ginsenoside Rg1 or a pharmaceutically acceptable salt thereof.
The active ingredient combination according to claim 5, wherein the clopidogrel or a pharmaceutically acceptable salt thereof, salvianolic acid B or a pharmaceutically acceptable salt thereof, and ginsenoside Rg1 or a pharmaceutically acceptable compound thereof An acceptable weight ratio of salt is 0.5-8: 0.5-6: 0.6-12, preferably 1-6: 0.5-4: 1-10, and more preferably 2-4: 1-3: 3-7.
A composition according to claim 1 or a kit according to claim 4 or an active ingredient combination according to claim 5 for use, comprising: (i) preparing an improved blood vessel Bleeding and microvascular occlusion reducing drugs during stent surgery; (ii) preparation of medicaments for improving bleeding and reducing microvascular occlusion during thrombolytic surgery; and / or (iii) preparation of medicaments for preventing and / or treating ischemia-reperfusion injury.
The use of a compound combination comprising (a) salvianolic acid B or a pharmaceutically acceptable salt thereof and (b) ginsenoside Rg1 or a pharmaceutically acceptable salt thereof, characterized in that The compound combination is used to prepare a pharmaceutical composition or formulation for use in: (i) improving bleeding and reducing microvascular obstruction during vascular stent surgery; (ii) improving bleeding and Reduce microvascular obstruction; and / or (iii) reduce the side effects of anticoagulants.
The use according to claim 8, wherein the weight ratio of salvianolic acid B or a pharmaceutically acceptable salt thereof to ginsenoside Rg1 or a pharmaceutically acceptable salt thereof is 0.5-6: 0.6-12 , Preferably 0.5-4: 1-10, more preferably 1-3: 3-7.
A method for simultaneously reducing the risk of bleeding and microvascular obstruction, characterized in that the method includes the step of administering to a desired subject a composition according to claim 1 or a kit according to claim 4.